Development of a CRISPR-SaCas9 system for projection- and function-specific gene editing in the rat brain

Abstract

A genome editing technique based on the clustered regularly interspaced short palindromic repeats (CRISPR)associated endonuclease Cas9 enables efficient modification of genes in various cell types, including neurons. However, neuronal ensembles even in the same brain region are not anatomically or functionally uniform but divide into distinct subpopulations. Such heterogeneity requires gene editing in specific neuronal populations. We developed a CRISPR-SaCas9 system-based technique, and its combined application with anterograde/ retrograde AAV vectors and activity-dependent cell-labeling techniques achieved projection-and function-specific gene editing in the rat brain. As a proof-of-principle application, we knocked down the cbp (CREB-binding protein), a sample target gene, in specific neuronal subpopulations in the medial prefrontal cortex, and demonstrated the significance of the projection-and function-specific CRISPR-SaCas9 system in revealing neuronal and circuit basis of memory. The high efficiency and specificity of our projection-and function-specific CRISPR-SaCas9 system could be widely applied in neural circuitry studies. Development of a CRISPR-SaCas9 system for projection-and function-specific gene editing in the rat brain. Sci. Adv. 6, eaay6687 (2020).

Combining CRISPR/Cas9 and brain imaging: from genes to molecules to networks

“…In addition to this, the intrinsically high intersubject variability in rs-fMRI, and differences in knockdown efficiency contributed to significant variance in our cohorts. Nevertheless, the variability of gene-editing efficiency was in line with previous in vivo brain studies [40, 41].…”

“…CRISPR/Cas9-editing overcomes these limitations, allowing gene-editing in adult and aged animals and avoiding compensatory changes occurring at early developmental stages. Since its discovery, only two studies have successfully applied CRISPR/Cas9 in the rat brain [40, 67], where gene-editing has been difficult to adapt. Rats are particularly advantageous for imaging studies due to the larger brain size and limited spatial resolution and sensitivity of preclinical scanners [68].…”

“…Despite these limitations, the potential of CRISPR/Cas9 is continuously expanding with novel nuclease variants being exploited [35][36][37]. Derived from Staphylococcus aureus, SaCas9 overcomes the packaging constraints of adeno-associated viral vectors (AAVs), allowing efficient CRISPR/Cas9 brain transfer [38][39][40][41][42].…”

Revisiting gene delivery to the brain: silencing and editing

“…262,264,265 The demonstration of the gene-editing potential of the CRISPR/Cas9 system has been demonstrated in the context of HD, [266][267][268][269] Alzheimer's disease (AD), 270,271 fragile X syndrome, 272 Parkinson's disease, 273,274 schizophrenia, 275 and neural stem cell gene repression, 276 among others, both in vitro and in vivo. The in vivo potential of the CRISPR/Cas9 system for brain applications has been confirmed in mice, 266,[268][269][270]272,276 rats 277,278 and monkeys 279 via intracerebral 266,[268][269][270][271][272]275,280 and intrathecal 275 administrations. Neurons, 270,272,277,[281][282][283] microglia, 272 neural stem/ progenitor cells, 276,283 and astrocytes 272 in animal models at embryonic, 280,284 fetal, and adult 270,…”

Genome editing technologies: CRISPR, LEAPER, RESTORE, ARCUT, SATI, and RESCUE

“…Although allelic correction and intergenerational transfer seem to be good practice, as stated before, unexpected results may be encountered due to the uncontrollable features of the CRISPR-Cas9 system. Another study on this subject is the deletion of the memory of rats with CRISPR (Sun et al, 2020[ 117 ]). This important claim will pave the way for the CRISPR system, to which we refer to rewritable codes, to erase and restore memory and perhaps load data that will at least restore people's daily routines in diseases such as Alzheimer's.…”